The transforming growth factor beta (TGF-β) superfamily is a large family of multifunctional proteins that regulate a variety of cellular functions, including cellular proliferation, migration, differentiation and apoptosis. TGF-β,∴the founding member of TGF-β family, has been shown to play a variety of roles ranging from embryonic pattern formation to cell growth regulation in adult tissues. TGF-β exerts its biological functions by signal transduction cascades that ultimately activate and/or suppress expression of a set of specific genes. Other TGF-beta family members include activins, inhibins and Bone Morphogenic Proteins (BMPs). BMP-mediated signal transduction is important for a variety of normal processes, including bone growth and the function of the nervous system, eyes and organs such as kidneys.
TGF-β family members generally initiate signal transduction by first binding to a receptor. TGF-β, for example, triggers its signal by first binding to its type II receptor, then recruiting and activating its type I receptors. The activated type I receptors then phosphorylate its intracellular signal transducer molecules, the Smad proteins (Heldin et al., Nature 390:465-471, 1997; Derynck et al., Cell 95:737-740, 1998). Similarly, BMP binds to a BMP serine/threonine transmembrane receptor protein kinases. The signals are further transduced from the receptors to the nuclei, resulting in altered patterns of gene expression. Signal transduction from BMP receptor to nuclei is known to involve Smad family proteins, certain of which become incorporated into transcriptional complexes and activate downstream genes.
Smads are receptor-activated, signal transducing transcription factors that transmit signals from TGF-β family receptors. Members of the Smad family of proteins have been identified based on homology to the Drosophilia gene Mothers against dpp (mad), which encodes an essential element in the Drosophilia dpp signal transduction pathway (see Sekelsky et al., Genetics 139:1347-1358, 1995; Newfeld et al., Development 122:2099-2108, 1996). Smad proteins are generally characterized by highly conserved amino- and carboxy-terminal domains separated by a proline-rich linker. The amino terminal domain (the MH1 domain) mediates DNA binding, and the carboxy terminal domain (the MH2 domain) associates with the receptor.
To date, eight Smad proteins have been identified and shown to participate in signal responses induced by TGF-β family members (see Kretzschmar and Massague, Current Opinion in Genetics and Development 8:103-111, 1998). These Smads can be divided into three subgroups. One group (Smads1, 2, 3, 5 and 8) induces Smads that are direct substrates of a TGF-β family receptor kinase. Another group (Smad 4) includes Smads that are not direct receptor substrates, but participate in signaling by associating with receptor-activated Smads. The third group of Smads (Smad6 and Smad7) consists of proteins that inhibit activation of Smads in the first two groups.
Smads have specific roles in pathways of different TGF-β family members. Among Smad proteins identified for TGF-β family members, Smad2 and Smad3 are specific for TGF-β signaling (Heldin et al., Nature 390:465-475, 1997). The activated Smad2 and Smad3 interact with common mediator Smad4 and translocate into nuclei, where they activate a set of specific genes (Heldin et al., Nature, 390:465-471, 1997). The TGF-β pathway uses the signal inhibitory proteins Smad6 and Smad7 to balance the net output of the signaling, as well as direct activation of Smad2 and/or Smad3. In the case of BMP-mediated signaling, following binding of a BMP to a BMP receptor, Smad1 and Smad5 are recruited to the receptor and phosphorylated. Once these proteins are phosphorylated, Smad1 and Smad5 form a complex with Smad4, and the complex translocates to the nucleus, resulting in activation of BMP-mediated gene transcription.
While Smad2 and Smad3 have intrinsic transactivation activity as transcription factors (Zawel et al., Mol Cell 1:611-617, 1998), studies have demonstrated that they activate specific gene expression largely through specifically interacting with other nuclear factors (Derynck et al., Cell 95:737-740, 1998). A specific TGF-β-mediated effect on a given cell type can be achieved by activating a specific Smad protein, resulting in alterations in expression of specific genes. The interplay or crosstalk of different signal transduction pathways is essential to provide balanced and integrated response to total signals to a given cell under given conditions. TGF-β-induced signaling has been found to crosstalk at the Smad level with Ras-mediated MAP kinase pathway and Jak/Stat pathway (Ulloa et al., Nature 397:710-3, 1999, Kretzschmar et al., Nature 389:618-22, 1997).
As noted above, TGF-β plays a role in the regulation of cell growth. TGF-β can be a growth stimulator or growth inhibitor, depending on the type or/and growth stage of the responding cells. As a potent negative epithelial cell growth regulator, TGF-β plays an important role in epithelial carcinogenesis (Cui et al., Cell, 86:531-542, 1996). TGF-β has been shown to cause cell growth arrest by inducing cyclin-dependent kinase inhibitors such as p15 and p21 (Hannon et al., Genes Dev. 9:1831-45, 1995), and a TGF-β type II receptor mutation that makes cells resistant to TGF-β leads to an enhancement of tumorigenic state of cells (Markowitz et al., Science 268:1336-8, 1995). Mutations in Smad genes have also been associated with cancer. Some colon cancers have found to carry mutations in tumor suppressor protein Smad2 (Eppert et al., Cell 88:543-552, 1996; Hata et al., Nature 388:82-87, 1997). It also has been shown that Smad4 is a tumor suppressor gene in human pancreatic carcinomas and perhaps in other tumors. Smad3 mutant mice develop metastatic colorectal cancer (Zhu et al., Cell 94:703-714, 1998), suggesting that Smad3 may play role in human colon cancer. In other contexts, TGF-β and TGF-β pathway members appear to play cell growth promoting roles. At early stages of carcinogenesis, TGF-β has been reported to act as a tumor promoter. At later stage, TGF-β can stimulate malignant progression. It has recently been demonstrated that TGF-β is directly involved in promoting malignancy following organ transplantation (Hojo et al., Nature 397:530-534, 1999). Thus, TGF-β can promote tumor cell invasion and metastasis, and methods for modulating TGF-β signaling could provide opportunities to develop effective cancer therapy.
Although certain aspects of TGF-β- and BMP-mediated signaling are understood, further knowledge of these signaling pathways is needed to facilitate the development of therapeutic agents that modulate such signaling. Accordingly, there is a need in the art for an improved understanding of the molecular mechanisms of TGF-β- and BMP-mediated signaling and for the development of agents that modulate such signaling. The present invention fulfills these needs and further provides other related advantages.